1. Field of the Presently Disclosed Subject Matter
The presently disclosed subject matter relates to compositions and methods pertaining to the modulation of ADAM 10. In particular, the presently disclosed subject matter relates to isolated and purified prodomain of ADAM 10, to truncations and mutations of prodomain polypeptides, and to modifications to stabilize prodomains for in vivo use. The presently disclosed subject matter further relates to the use of prodomains in cellular assays, and to the use of prodomains for treatment of diseases such as cancer, neurological disorders, asthma, and allergic responses.
2. Description of the Related Art
ADAM 10 is a member of the a disintegrin and metalloproteinase (ADAM) family (1) that includes enzymes such as TACE (ADAM 17), ADAM 8, and ADAM 9. In total, for humans, there are 33 ADAM family members. The ADAM proteins comprise a prodomain that is important for proper folding and transport of the enzyme through the cell, a catalytic domain containing a typical HEXXH motif, a disintegrin domain, that is used to interact with integrins, a cysteine rich region that is believed to be important for substrate recognition, a transmembrane domain, and a cytoplasmic tail that is involved in signaling events.
Members of the ADAM family are known to cleave type I and type II single membrane spanning proteins from cells to generate soluble mature proteins that have varying physiological roles (2). For example, TACE is known to generate soluble epidermal growth factor (EGF) ligands such as TGF-alpha, amphiregulin, and HBEGF (3). Similarly, ADAM 10 activity generates soluble proteins including, but not limited to, EGF ligands, EGF and betacellulin (3), Notch (4), amyloid precursor protein (5), ephrins (6), cadherins (7), protocadherins (8), chemokines such as CXCL16 and CX3CL1 (9), HER2 (10), AXL (11), and CD23, a low affinity receptor for IgE (12). Disruption of ADAM 10 activity has been shown to decrease the level of soluble non-amyloidogenic APP both in vivo and in cell based assays (13), suggesting that maintaining ADAM 10 activity may play a protective role in Alzheimers disease for normal processing of soluble APP-α. In contrast, excess ADAM 10 activity may promote cell growth in cancer proliferation assays due to enhanced production of soluble epidermal growth factor (EGF) ligands (14).
Inhibition of TACE activity is correlated with beneficial effects in a tumor cell proliferation assay (15). The mechanism for this inhibition of tumor cell proliferation is believed to be through prevention of EGF ligand release. For example, EGF ligands such as TGF-alpha, amphiregulin, HB-EGF, EGF and betacellulin, once released, are capable of activating the EGF receptor, which in turn leads to cancer proliferation (3). Similar to TACE, ADAM 10 promotes production of soluble EGF ligands such as EGF and betacellulin, however, unlike TACE, ADAM 10 also generates soluble Notch (4) and AXL (11) that are known promoters of tumor cell proliferation.
In addition to EGF ligands, ADAM 10 also generates soluble CD23 (12). Release of CD23 promotes allergic responses through activation of IgE (16). Metalloproteinase inhibitors have been shown to block CD23 shedding and prevent allergic responses in both in vitro and in vivo assays (17).
Accordingly, the ability to specifically modulate ADAM 10 activity would be useful to study the biological functions of the protein, and for the treatment of disorders including cancer, neurological disorders, asthma, and allergic responses. Unfortunately, existing small molecule inhibitors are not specific for ADAM 10 activity. For example, hydroxamates developed by GSK inhibit both ADAM 10, as well as other members of the matrix metalloproteinase family (9). Inhibitors disclosed by Incyte also inhibit MMPs, and possibly other ADAM family members (10). Such non-specific inhibition often leads to unwanted side effects, and in this case has prevented the compounds from being developed into pharmaceutical drugs (18).
Another approach to this problem is to use ADAM protein prodomains as selective inhibitors. ADAM family members are expressed as zymogens with the prodomains maintaining the enzymes in a latent state. However, while isolated prodomains have been shown to inhibit the proteolytic activity of ADAM family proteins in vitro, not all prodomains are good inhibitors. For example, the prodomain of TACE suppresses the activity of its catalytic domain with a Ki of 50 nM (19), but the prodomain is only a weak (high micromolar) inhibitor of a TACE construct consisting of both the catalytic and disintegrin domains. Therefore, the prodomain is unlikely to negatively affect TACE activity in vivo, as the catalytic and disintegrin domains are both retained in membrane bound TACE. Furthermore, for those prodomains that do inhibit ADAM activity, they have never been tested to determine if they are specific inhibitors of their respective ADAMs.
Accordingly, there is a need in the art for selective modulators of ADAM proteases to study the biological functions of the proteins and to treat diseases such as cancer, neurological disorders, asthma, and allergic responses.